摘要:微生物在众多的自然和人工生态系统中发挥着核心的作用，但能够被培养分离的微生物在大部分生态系统中只占极少一部分，极大地限制了人们对微生物组成、功能及其潜在应用的认识。分子生物学方法，尤其是高通量测序技术应用到微生物生态学研究中，为认识微生物多样性、群落结构组成及其生态功能提供了有利手段。高通量测序作为一种新兴的免培养分子生物学技术，具备检测快速、准确、信息全面丰富等特点。随着高通量测序技术的不断升级换代，测序通量、读长和准确度的不断提升以及成本的大幅下降，该技术在过去十几年间被迅速应用于土壤、水体和肠道等微生物区系的研究中。本文简述了基于高通量测序技术的PCR产物测序技术和宏基因组学测序技术的原理、发展历程、数据分析方法与应用，以及宏基因组学测序技术在病毒学领域的应用，以期为微生物分子生态学研究提供参考。
关键词:高通量测序/
微生物分子生态学/
PCR产物测序/
宏基因组学/
病毒
Abstract:Microorganisms play essential roles in natural and artificial ecosystems. However, only a small portion of microorganisms can be cultured and isolated in most ecosystems, which greatly limits our understanding of microbial community composition, ecological function and their potential applications. The novel molecular techniques, especially high-throughput sequencing, have provided advantages for further exploring the diversity, composition and ecological functions of microbial community. High-throughput sequencing is a culture-independent technology capable of deep, rapid and accurate detection of genetic information. With the update of the high-throughput sequencing technology, the sequencing throughput, read length and accuracy have been dramatically improved, and the cost has greatly declined. During the past decade, high-throughput sequencing technology has been rapidly applied in the microbial ecological studies with various types of samples such as soil, water and gut microbial communities. Amplicon and metagenomic sequencing are most widely used strategies for environmental samples. Amplicon sequencing refers to sequencing the PCR products of target gene fragments amplified with specifically designed primers. The 16S rRNA gene of prokaryotes and the 18S rRNA and ITS genes of eukaryotes are most commonly used marker genes to conduct microbial taxonomic analysis. In addition, functional genes such as nirK, nirS and nosZ genes of denitrifying bacteria, amoA gene of ammonia-oxidizing bacteria, and ureC gene of urea hydrolytic bacteria are frequently adopted to study the diversity of functional microorganisms. In metagenomics, sequencing is performed on the genomic DNA directly extracted from environmental samples therefore avoiding the bias from PCR. Theoretically this method can provide a representation of all genomes in the sample and can be used for fully exploring the genetic diversity, functional potentials and metabolic pathways of both cultured and uncultured microorganisms. Metagenomic sequencing technology has been applied in the field of medical diagnosis, human health, biological energy, environmental restoration and agricultural ecology, etc. and has provided us new insights into taxonomic diversity, ecological function, evolutionary succession and interaction in the complex microflora. The application of metagenomic sequencing technology to the field of virology is referred as viral metagenomics. Viruses are not only related to various diseases of crop, animal and human, but also indispensable in the natural ecosystems, and play an important role in regulating host diversity and community succession, mediating gene transfer between microbes, and promoting global biogeochemical cycles. Viral metagenomics has recently gained momentum in application in the field of environmental science to reveal the genetic diversity, explore the novel species of viruses, and investigate their interactions with environmental factors.
Key words:High-throughput sequencing/
Microbial ecology/
Amplicon sequencing/
Metagenomics/
Virus
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